Method for the purification of a glycan and/or a glycoconjugate by chromatography using a stationary phase comprising cotton

Abstract

A method of purifying a glycan and/or a glycoconjugate comprising the steps of: (a) providing a stationary phase that comprises cotton; (b) applying a glycan and/or glycoconjugate-containing sample to the stationary phase; (c) washing the stationary phase with a first solvent; and (d) eluting the glycan and/or glycoconjugate from the stationary phase with a second solvent. A kit for purifying a glycan and/or glycoconjugate, the kit comprising: a stationary phase comprising cotton; and instructions for purifying a glycan and/or glycoconjugate according to the disclosed method.

The invention claimed is: 
     
       1. A hydrophilic interaction liquid chromatography solid phase extraction method of purifying a glycan and/or glycoconjugate, the method comprising:
 applying a glycan and/or glycoconjugate-containing sample comprising an organic solvent to a stationary phase that is cotton wool fibers; 
 washing the stationary phase with a first solvent which is a solvent mixture comprising between 70% volume per volume (v/v) and 95% v/v organic solvent and between 0.1% v/v to 3% v/v acid in water; and 
 eluting the glycan and/or glycoconjugate from the stationary phase with a second solvent, wherein the second solvent is one of water, dimethylsulfoxide, or dimethylformamide. 
 
     
     
       2. The method of  claim 1 , wherein the organic solvent of the glycan and/or glycoconjugate-containing sample at least one of acetonitrile, methanol, ethanol, propanol, isopropanol, butanol, or tetrahydrofuran. 
     
     
       3. The method of  claim 1 , wherein the organic solvent of the first solvent comprises at least one of acetronitrile, methanol, ethanol, propanol, isopropanol, butanol, or tetrahydrofuran, and the acid comprises at least one of trifluoroacetic acid, formic acid, acetic acid, pentafluoropropionic acid, or heptafluorobutyric acid. 
     
     
       4. The method of  claim 1 , wherein the second solvent comprises more polar solvent than the first solvent. 
     
     
       5. The method of  claim 1 , wherein the glycoconjugate comprises at least one of a glycoprotein, glycopeptide or glycolipid. 
     
     
       6. The method of  claim 1 , wherein the glycan comprises a N-glycan. 
     
     
       7. The method of  claim 1 , wherein the stationary phase is re-usable. 
     
     
       8. The method of  claim 1 , wherein the stationary phase contains about 500 μg of the cotton wool fibers. 
     
     
       9. The method of  claim 1 , further comprising performing mass spectrometric analysis on the eluted glycan and/or glycoconjugate. 
     
     
       10. The method of  claim 1 , further comprising glycosylation profiling at a glycopeptide level of the eluted glycopeptides. 
     
     
       11. The method of  claim 1 , wherein the stationary phase is held in an open ended vessel. 
     
     
       12. The method of  claim 1 , wherein the method is utilized to one of extract glycans after PNGase F treatment of glycoproteins, extract glycans after fluorescent labelling by reductive amination, or enrich N-glycopeptides from proteolytic digests. 
     
     
       13. The method of  claim 1 , including, a kit for purifying a glycan and/or glycoconjugate, the kit comprising:
 a stationary phase that is cotton wool fibers; and 
 
       instructions for purifying a glycan and/or glycoconjugate according to the method of  claim 1 . 
     
     
       14. The method of  claim 2 , wherein the organic solvent of the glycan and/or glycoconjugate-containing sample is between 70% and 88% v/v acetronitrile in water. 
     
     
       15. The method of  claim 3 , wherein the first solvent mixture comprises between 75% and 90% v/v organic solvent and between 0.1% and 1% v/v acid in water. 
     
     
       16. The method of  claim 5 , wherein the glycopeptide is an IgG glycopeptide. 
     
     
       17. The method of  claim 16 , wherein the IgG glycopeptide is a tryptic IgG Fc N-glycopeptide. 
     
     
       18. The method of  claim 9 , wherein the mass spectrometric analysis is MALDI-TOF MS detection. 
     
     
       19. The method of  claim 11 , wherein the open-ended vessel is one of a pipette, a multi-channel pipette or a pipette tip. 
     
     
       20. The method of  claim 11 , wherein the open-ended vessel is a microtip.